Abstract
Objective
To improve the transformation efficiency of Corynebacterium glutamicum cells with heterogenous plasmid DNA and single-strand DNA (ssDNA) using a methodology based on electro-transformation.
Results
A semicomplex hypertonic medium was selected with addition of glycine and dl-threonine to weaken cell walls and addition of Tween 80 and isonicotinic acid hydrazide to increase cytoplasmic membrane fluidity. Their contents were optimized by response surface methodology. Cell growth, electro-transformation buffer, and transformation protocol were also optimized. Temporary heating inactivation of the host restriction enzyme showed a significant effect. Finally, a high transformation efficiency of 3.57 ± 0.13 × 107 cfu/μg DNA of plasmid and 1.05 × 106 StrR cfu per 109 viable cells with a ssDNA was achieved.
Conclusion
The results shed light on the application in functional genomics and genome editing of C. glutamicum.
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Acknowledgments
This work was supported by the Fundamental Research Funds for the Central Universities (JUSRP51306A & JUDCF13008 & JUSRP51402A), the Priority Academic Program Development of Jiangsu Higher Education Institutions, and Program of Introducing Talents of Discipline to Universities (No. 111-2-06).
Supplementary data
Supplementary Table 1—CCD matrix of four variables in coded units and the experimental and predicted values of transformation efficiency.
Supplementary Table 2—ssDNA sequence with different length targeted to modify rpsL gene.
Supplementary Table 3—Significance of the regression coefficients for the response surface model.
Supplementary Fig. 1—Mathematical model of the transformation efficiency predicted by response surface methodology.
Supplementary Fig. 2—Three-dimensional response surface and contour plots of the interactive effect between every two factors of Gly, dl-Thr, Gly and Tween 80 on the ETE of C. glutamicum. Actual factors of (A–F) are set at 0 in coded levels, respectively.
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Ruan, Y., Zhu, L. & Li, Q. Improving the electro-transformation efficiency of Corynebacterium glutamicum by weakening its cell wall and increasing the cytoplasmic membrane fluidity. Biotechnol Lett 37, 2445–2452 (2015). https://doi.org/10.1007/s10529-015-1934-x
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DOI: https://doi.org/10.1007/s10529-015-1934-x